1. Field of the Invention
The present invention relates to a method for regenerating the oxygen-binding ability of hemoglobin, which has been lost through oxidation, in a hemoglobin-vesicle suspension serving as an oxygen infusion (oxygen carrier), thereby maintaining its oxygen-transporting ability.
2. Description of the Related Art
The vesicle suspension including hemoglobin-vesicles can be widely used in the medical and pharmaceutical fields. In particular, the vesicular suspension, if various additives are added thereto, can be used as a blood substitute in clinical treatments.
The currently used transfusion systems for injecting human blood into a blood vessel have the following problems.
1) Infection with hepatitis and AIDS viruses may occur.
2) The storage limit of erythrocytes is 3 weeks.                3) Due to coming of aged society, the ratio of the aged patients/all patients requiring transfusion increases, whereas the total number of healthy blood donors is decreasing.        
4) Contamination may occur during storage.
5) Human blood cannot be given to patients who refuse the human-blood transfusion for religious reasons.
6) The system cannot satisfy emergency needs in disaster situations.
Under these circumstances, there are great demands for an blood substitute which is available anytime and which replaces all blood types. Electrolyte transfusions and coloidal transfusions have hitherto been widely used as the blood substitution. However, these substitutes are devoid of the most important function of blood. More specifically, they do not substitute the function of erythrocytes for transporting oxygen. Hence, it has been demanded to develop a substance (oxygen transfusion) for substituting the oxygen-transporting function.
Oxygen infusion using hemoglobin (human hemoglobin, bovine hemoglobin, and recombinant hemoglobin) having an oxygen association/dissociation function have been developed. Furthermore, clinical tests have been conducted on intramolecular cross-linked hemoglobin, water-soluble polymer conjugated hemoglobin, and intermolecularly cross-linked and polymerized hemoglobin in Europe and the United States. However, it has been pointed out over time that various types of side effects are produced due to this non-cellular form of hemoglobin. Based on the clinical tests, it became apparent that the encapsulated hemoglobin, so-called cellular-type hemoglobin plays an important role.
It was found that a biologica component, phospholipid, forms the vesicle or a liposome structure by itself. In addition, Djordjevich and Miller (Fed. Proc. 36, 567, 1977) started studies on the hemoglobin-vesicle using a liposome formed of phospholipid/cholesterol/fatty acid. Since then, several groups, including the group of the present inventors, have conducted extensive studies of so called the hemoglobin-vesicle. The hemoglobin-vesicle has the following advantages.
1) It can be used as it is without modification of molecular hemoglobin.
2) Values of viscosity, colloidal osmotic pressure, and oxygen affinity can be arbitrarily adjusted.
3) Retention time in blood can be extended.
4) Various types of additives can be included in an aqueous phase within the vesicle at high concentrations.
Among these advantages, the advantage 4) is particularly important in the present invention. The present inventors originally established an efficient method for preparing the hemoglobin-vesicles. As a result, they obtained a hemoglobin-vesicles having physical properties extremely close to those of blood. The fact that the hemoglobin-vesicles transfusion has excellent oxygen transporting ability has been confirmed in animal administration tests (Tsuchida ed. Blood Substitutes Present and Future Perspectives, Elsevier, Amsterdam, 1998).
Hemoglobin contains four heme groups. When heme iron is ferrous iron (Fe2+), oxygen can be reversibly bound to the ferrous iron. However, when the heme iron is in the oxidation state of ferric iron (Fe3+) (called methemoglobin), oxygen cannot bind to the ferric iron. In addition, the oxygen-bound hemoglobin gradually releases a superoxide anion and changes into methemoglobin. Furthermore, the superoxide anion acts as an oxidizing agent to accelerate production of methemoglobin. In erythrocytes, there are a methemoglobin reducing system and an active oxygen removal system, which prevent the content of methemoglobin from increasing, whereas, in the hemoglobin-vesicle employing purified hemoglobin, these enzymatic systems are all eliminated in a purification step. Therefore, hemoglobin is oxidized during storage and after administration (to a body), lowering the oxygen-transfer ability. To suppress the oxidation reaction, the following methods are presently used: a method of purifying hemoglobin under mild conditions which will not inactivate the enzyme (Ogata et al. Artificial Blood 2, 62-66, 1994); a method wherein a reducing agent (glutathione, homocystine, and/or ascorbic acid) as well as an enzyme (catalase and/or superoxide dismutase) which eliminate active oxygen are added (Sakai et al., Bull, Chem, Soc. Jpn., 1994); and a method wherein metohemoglobin contained in the vesicle is reduced by adding methylene blue into the vesicle membrane, which serves as an electron transfer carrier and allows electrons transfer from NADH in the outer aqueous phase into the vesicle (Takeoka et al., Bull, Chem, Soc, Jpn, 70, 1171-1178, 1997).
On the other hand, a phenomenon where methemoglobin or cytochrome C is reduced by light irradiation has been reported, for the first time, by Vorkink and Cusanovich (Photochem. Photobiol. 19, 205-215, 1974), independently of the oxygen transfusion. In addition to this report, a phenomenon is found where a reduction reaction is also advanced by light irradiation in myoglobin and cytochrome oxidase etc. Since then, the photoreduction of a heme protein has been investigated by many biochemists (Kitagawa & Nagai, Nature, 281, 503-504, 1979; Kitagawa et al., J. Sm. Chem. Soc. 106, 1860-1862, 1984; Morikis et al., J. Biol. Chem. 265, 12143-22145, 1990; Sage et al., J. Chem. Phys. 90, 3015-3032, 1989; Gu et al., J. Am. Chem. Soc., 115, 4993-5004, 1993; Pierre et al., Eur. J. Biochem, 124, 533-537, 1982; Bazin et al., Eur. J. Biochem, 124, 539-544, 1982).
Furthermore, the following phenomenon is also known. When an oxidized flavin is added together with various types of sacrificial reagents (electron donor) to a methemoglobin solution and visible light of about 450 nm is directed to the resultant solution, a reduced-type flavin is generated, which in turn reduces methemoglobin (Yubisui et al., J. Biol. Chem. 255, 11694-11697, 1980; Everse, Methods Enzymol. 231, 524-536, 1994).
The aforementioned conventional method for reducing the oxidized-hemoglobin-vesicle has the following problems.
When blood is used as a raw material, inactivation of viruses must be primarily performed in the purification step of hemoglobin. Heating of hemoglobin is desirably performed at 60° C. for 10 hours, in the same manner as in albumin preparation. However, in the heating step, the methemoglobin-reducing enzymatic system inherently present in erythrocytes is also denatured and inactivated. The activity of the enzymatic system can be retained if the purification is performed under mild conditions, for example, in accordance with a hypo-osmotic hemolysis method. In this case, oxidation of the resultant hemoglobin-vesicle can be suppressed. However, inactivation of viruses cannot be attained. In addition, the enzymatic system is chemically labile, so that the activity of the enzymatic system decreases during storage.
Alternatively, if a relatively mild reducing agent such as glutathione or homocysteine is included in the hemoglobin-vesicle as mentioned above, heme iron previously oxidized into ferric iron is reduced into ferrous iron. Therefore, the oxidation reaction is suppressed as a whole. These reducing agents are oxidized slightly and gradually inactivated even if methemoglobin is not present. It has been therefore desired to develop a system for reducing methemoglobin to hemoglobin only when the content of methemoglobin increases.
Furthermore, as described above, it has been reported that a reduction reaction is started by applying light to a dilute methemoglobin solution, as mentioned above. However, this phenomenon occurs with an extremely low efficiency in a homogeneous solution system.